The origin of eukaryotes was marked by the emergence of entirely new subcellular systems
as well as novel components in preexisting systems. Deciphering the evolutionary history
and the ultimate provenance of these systems and components, which were long considered
to be quintessential features of eukaryotes, has considerably advanced in the past
decade as a result of the growing genomic data and concomitant comparative genomics
analysis 1]-4]. In this regard, we have had a long-standing interest in understanding the origins
of eukaryotic innovations relating to ribosome biogenesis and the translation machinery
5],6]. In several cases, we have been able to identify prokaryotic homologs of what previously
seemed to be purely eukaryote-specific components in these systems. Recognition of
these prokaryotic versions has helped clarify the precise evolutionary trajectories
by which these components were recruited to the eukaryotic ribosome biogenesis/translation
apparatus. Moreover, these studies have also often helped predict the potential biochemical
roles of several poorly understood components in these systems by exploiting the contextual
information available in prokaryotic genomes 5],6].
In this study we present an investigation of the conserved eukaryotic regulator of
translation initiation CDC123 and it homologs. CDC123 was first identified over 30
years ago in a screen for temperature-sensitive mutations that blocked cell proliferation
in rat fibroblast cells 7]. This was attributed to a cell-cycle related function arising from its apparent functional
interaction with checkpoint proteins chf1/chf2 8], which are active in triggering mitosis entry 9]. Conditional mutants in the Saccharomyces cerevisiae cognate were shown to result in increased heat sensitivity, whereas CDC123 null mutants
were inviable 8]. Further investigation of these phenotypes pointed to a role in translation as it
was observed that CDC123 specifically regulates the abundance of the eukaryotic translation
initiation eIF2 complex 8],10], and binds one of its components yeast GCD11 or its human ortholog eIF2? 11],12] in the cytoplasm. To date its orthologs have only been reported from eukaryotes,
where it is widely distributed across all major lineages of the eukaryotic tree. This
phyletic pattern, together with its essentiality in yeast, suggest that CDC123 might
indeed be a conserved regulator of translation. However, despite over three decades
of research on CDC123, its precise role in translation or cell-cycle regulation remains
unclear. Given these observations and the mounting evidence suggesting possible links
between CDC123 and a variety of human disease states including breast cancer 13], type II diabetes 14], and COPD 15], we sought to apply state-of-the-art methods in comparative sequence and genome analysis
to better understand the biochemical roles of CDC123.
We show that CDC123 defines a novel, highly-derived clade of the ATP-grasp superfamily
of enzymes 16],17]. We define the conserved sequence and structure features of this clade of ATP-grasp
proteins and predict they are likely to catalyze protein modifications by the formation
of amide/peptide-like linkages in an ATP-dependent manner. In addition, we identify
the first bacterial homologs of CDC123 where they are often found as part of the type
VI secretion systems (T6SS) that deliver polymorphic toxins 18],19]. Further, we show that this clade of ATP-grasp domains additionally includes two
previously unknown, related prokaryotic families with potential roles in distinct
biological conflict systems 20]-22]. Finally, we present evidence that the eukaryotic CDC123s might have been derived
from an ancestral bacterial conflict system in the stem eukaryote and recruited for
a role in protein modifications, including in context of translation initiation.
CDC123 contains an ATP-grasp module and has several distinct bacterial homologs
To better characterize CDC123, we initiated iterative sequence profile searches with
CDC123 orthologs known from prior studies as queries using the PSI-BLAST and JACKHMMER
programs (see Methods). Beyond the previously-identified homologs in animals, plants,
fungi, and stramenopiles 8], we detected eukaryotic orthologs spanning all other major branches of the eukaryotic
tree. For example, a search initiated with the yeast CDC123 recovered orthologs from
the apicomplexans, kinetoplastids, parabasalids and diplomonads within 2 iterations
with PSI-BLAST (Additional file 1). Concomitantly, these searches also recovered sequences from diverse bacterial and
viral lineages. For example, the above search recovered sequences from the ?-proteobacteria
Erwinia chrysanthemi (gi: 654084322, iteration: 1; e-value 6e-6) and Legionella pneumophila (gi: 652968979; iteration: 2; e-value: 2e-08), the planctomycete Zavarzinella formosa (gi: 521962559, iteration: 2; e-value: 8e-09), and the nucleocytoplasmic large DNA
virus (NCLDV) 23] Megavirus Iba (gi: 448825053; iteration: 2; e-value 1e-11).
Reverse searches initiated with these bacterial sequences recovered their eukaryotic
counterparts in initial iterations, then recovered several prokaryotic sequences either
unannotated or annotated as containing the “Domain of Unknown Functionâ€, DUF4343 24], before finally recovering sequences containing known ATP-grasp domains, typically
those most-closely related to RimK and RimK-like ATP-grasp families 16]. For example, a search initiated with the bacterial CDC123 homologue from Lentisphaera araneosa (gi: 494490064) recovers a sequence annotated as containing the DUF4343 domain from
bacterium Deinococcus pimensis (gi:653301678; iteration: 4; e-value: 4e-3), a sequence from the bacterium Pseudomonas aeruginosa with no previously identified domain (gi: 489255144; iteration: 6; e-value: 4e-05),
and a RimK-like ATP-grasp fold 25] domain from Herpetosiphon aurantiacus (gi: 501142781; iteration: 8; e-value: 2e-04). We further confirmed these results
using an HMM-(Hidden Markov Model) based method for detecting distant homology. HMMs
constructed with the CDC123 sequences as seeds were searched against a library of
pre-constructed HMMs based on Pfam domain definitions 26] and solved PDB (Protein Data Bank 27]) structures with the HHpred program. The strongest relationship detected in these
searches was consistently with the Pfam DUF4343 domain, followed by detection of other
ATP-grasp families and structures including the Pfam DUF3182 domain, a heretofore
unrecognized member of the ATP-grasp fold sharing conserved features and general sequence
affinity with a clade of ATP-grasp enzymes including the carbamoyl phosphate synthases
and BtrJ-like butirosin biosynthesis enzymes (Additional File 1). For example, a HMM constructed using the yeast CDC123 sequence as a seed detected
a significant relationship with the DUF4343 Pfam domain (p-value: 5.7e-10), the RimK-like
ATP-grasp domain (p-value: 9.8e-07), and the RimK structure from Thermus thermophilus (PDB: 3VPD; p-value: 6.7E-06). However, in terms of reciprocal recovery in sequence-similarity
searches and sequence similarity- and length-based clustering with the BLASTCLUST
program (see Methods), none of CDC123 and its newly identified homologs overlapped
with any previously known ATP-grasp families 28],16]. Together, these results strongly suggest that these sequences define a previously-unrecognized
clade of ATP-grasp-like proteins, which includes the CDC123, DUF4343-containing proteins,
and several additional unannotated prokaryotic proteins.
Distinctive features of the novel ATP-grasp clade and identification of three distinct
families within it
The catalytic module of the ATP-grasp superfamily is constructed from two distinct
domains: the N-terminal RAGNYA domain and the C-terminal protein kinase/PIPK-like
domain 29]-31]. In addition to this catalytic module, most members of the ATP-grasp superfamily
are fused at the N-terminus to the pre-ATP-grasp domain 16]. The position of the catalytic residues are typically conserved across the superfamily
and include: 1) a positively-charged residue, typically a lysine, found in the linker
region connecting the pre-ATP-grasp domain with the RAGNYA domain, 2) an additional
positively-charged residue, again typically a lysine, found near the C-terminal end
of the second strand of the RAGYNA domain, 3) an acidic residue, typically an aspartate,
located in the central region of the fourth strand of the protein kinase-like domain,
and 4) a conserved motif typically of the form ExN (where ‘x’ is any residue) located
at the C-terminus of the fifth and final conserved strand of the protein kinase domain
16]. Additionally, a large, monophyletic clade of ATP-grasp superfamilies, including
most peptide/amide bond-forming ligases members, contain a conserved arginine residue
in the first strand of the protein kinase-like domain 16] (Additional File 1).
Comparison of the features of the newly-identified clade to the above-described ATP-grasp
template revealed considerable concordance (for example: K104, D233, and D246xN248
correspond to features 2-4 listed above in the human CDC123 protein). However, striking
differences were observed: 1) In other ATP-grasp families the loop between strands
2 and 3 of the RAGNYA domain is well-conserved in terms of length (usually 9 amino
acids) and harbors a conserved ssxGbGl motif (where ‘s’ is any small residue, ‘b’ is any big residue, and ‘l’ is any aliphatic residue) 16]. However, in this novel clade this loop displays considerable length diversity and
lacks the above sequence motif. 2) The lysine typically observed in the linker region
between the pre-ATP-grasp domain and the RAGNYA domain is consistently absent in all
members of this newly detected clade (Figure 1). Instead, they display a distinct conserved lysine/arginine in the above-stated
loop, just downstream of the absolutely conserved lysine in strand 2 (Figure 1). This loop region is spatially positioned in close proximity to the active site
28]. Hence, we predict the conserved lysine/arginine from this loop likely acts as a
secondarily-acquired, compensatory residue that functions in lieu of the conserved
lysine from the pre-ATP-grasp-RAGNYA linker region. Indeed, these shared features
strongly support the monophyly of this clade of ATP-grasp enzymes and we propose naming
this novel clade the R2K ATP-grasp clade, for RAGNYA-containing 2 lysines (K).
Figure 1. Multiple sequence alignment of three families of R2K ATP-grasp modules with known
ATP-grasp structures. Proteins are labeled with their species abbreviations and GenBank index numbers along
with gene names for human and viral homologs. PDB identifiers, colored in orange,
are given in lieu of gene names where applicable. Secondary structures are depicted
above alignment with loop regions shown as lines, ?-strands (S1-S9) shown as green
arrows and ?-helices shown as orange cylinders. The coloring of the alignment is based
on 75% consensus shown below the alignment, using the following scheme: h, hydrophobic
(shaded in yellow); s, small (shaded in light green); l, aliphatic (shaded in yellow);
p, polar (shaded in light blue); +, positively charged; b, big (shaded in gray); a,
aromatic (shaded in yellow); c, charged (shaded in purple). Predicted catalytic residues
are colored in white and shaded in red. Species abbreviations: Acas, Acanthamoeba castellanii; Achl, Arthrobacter chlorophenolicus; Adel, Auricularia delicata; ApMV, Acanthamoeba polyphaga moumouvirus; Asp., Acaryochloris sp.; BPMyrna, Mycobacterium phage Myrna; BPRSL1, Ralstonia phage RSL1; Bsp., Brenneria sp.; Cfla, Chthoniobacter flavus; Cmin, Chamaesiphon minutus; Einv, Entamoeba invadens; Elat, Eutypa lata; Esp., Eggerthella sp.; Fnec, Fusobacterium necrophorum; Gint, Giardia intestinalis; Gsp., Geitlerinema sp.; Hsap, Homo sapiens; Krac, Ktedonobacter racemifer; Lbic, Laccaria bicolor; Llon, Legionella longbeachae; Lsab, Lachnoanaerobaculum saburreum; Lsp., Labrenzia sp.; Mmar, Microscilla marina; Mxan, Myxococcus xanthus; Ngru, Naegleria gruberi; Nvec, Nematostella vectensis; PVs, Pithovirus sibericum; Pamy, Pseudomonas amygdali; Pmar, Perkinsus marinus; Pmar, Planctomyces maris; Scer, Saccharomyces cerevisiae; Smob, Streptomyces mobaraensis; Ssp., Streptomyces sp.; Ster, Sebaldella termitidis; Tazo, Treponema azotonutricium; Tbry, Treponema bryantii; Tvag, Trichomonas vaginalis. Other abbreviations: GS, glutathione synthase; BC, biotin carboxylase.
To further understand the relationships within the R2K clade, we clustered its representatives
using sequence similarity- and length-based scoring parameters with the BLASTCLUST
program (Additional file 1). The results identified three distinct families: 1) the CDC123 or R2K.1 family consisting
of the pan-eukaryotic CDC123-like proteins, close homologs in certain NCDLVs infecting
microbial eukaryotes, and bacterial versions from ?-, ?-, and ?-proteobacteria, planctomycetes,
lentisphaerae, and firmicutes; 2) the R2K.2 family sporadically present across many
bacteria and a few bacteriophages, typically annotated as matching the Pfam DUF4343
model; 3) the R2K.3 family with a similar phyletically wide, yet sporadic, distribution
in bacteria with rare archaeal representatives. The R2K.3 family is often misannotated
as a “membrane proteinâ€, typified by the sce1853 protein in Sorangium cellulosum. Each of the families is clearly distinguished from the other by the spacing of the
second conserved lysine with respect to the absolutely conserved lysine in strand
2 of the RAGNYA domain (Figure 1). A subset of the families or members within each family might show certain peculiarities:
the eukaryotic versions of the CDC123 family are often characterized by large, variable,
low complexity inserts within the catalytic module predicted to be structurally disordered.
The pre-ATP-grasp domain is well-conserved in the R2K.3 family but is rapidly diverging
in the CDC123 and the R2K.2 families. The R2K.3 family is further distinguished by
an unusual constellation of conserved residues in the final strand of the protein
kinase/PIPK-like domain of the ATP-grasp module, where it contains an ExGD motif instead
of the standard ExN motif (Figure 1). While the N residue is, on occasion, substituted for distinct polar residues, the
migration of the residue one position downstream has not, to our knowledge, previously
been observed in the ATP-grasp superfamily.
Evolutionary history of the R2K clade ATP-grasp enzymes
Despite their distinctive features, the fusion to the pre-ATP-grasp domain points
to the R2K clade being deeply nested within the previously defined tree of ATP-grasp-like
modules 16] (Additional File 1). Moreover, the presence of the conserved arginine residue in the first strand of
the protein kinase/PIPK-like domain of the ATP-grasp module (part of the conserved
ExR motif in S5 of Figure 1) suggests that the R2K clade specifically belongs to a larger assemblage within the
superfamily that is almost entirely comprised of ligases catalyzing peptide-like linkages
16]. This assemblage includes the ATP-grasp enzymes catalyzing the formation of such
bonds in cofactors (e.g. glutathione), antibiotics 32],33], peptidoglycan 34],35], siderophores 36], the biosynthesis of lysine (LysX), and catalyzing polyglutamyl and polyglycinyl
modification of cofactors and proteins like ribosomal protein S6 and tubulin 37],38]. The majority of these families appear to have first radiated in the bacteria 16]. Similarly, all three families of the R2K clade have a bacterial presence, with the
eukaryotic CDC123s nested within the bacterial diversification of this clade in a
phylogenetic tree (Figure 2). These observations suggest the R2K clade first emerged in bacteria followed by
initial diversification into three distinct families. Additionally, the phyletic patterns
of bacterial versions and their relationships in the phylogenetic tree (Figure 2, Additional File 1) strongly suggest horizontal gene transfer (HGT) as the key theme in their evolution.
Figure 2. Evolutionary relationship of three families of the R2K ATP-grasp module shown to the
left and conserved contextual associations including operonic organizations and domain
architectures are provided on the right. Tree nodes supported by bootstrap 75% are shown. Proteins are denoted by their GenBank
index numbers and their complete species names and colored according to their lineages:
bacterial in blue, viral in green, amoeboazoan in orange, Naegleria in purple, fungal in red. Conserved gene neighborhoods are depicted as boxed, labeled
arrows with the arrowhead pointing to the C-terminus of the protein. Genes known to
be part of the T6SS are shaded in gray, including the “T6SS.unk†gene containing a
domain of unknown function in the secretion system. Conserved domain architectures
are depicted as adjoining, labeled shapes.
Two distinct versions of the CDC123 (R2K.1) family are found in eukaryotes. The phyletic
patterns suggest that the classical CDC123 orthologs, typified by relatively short
average branch terminal lengths (Figure 2), were likely to have been present in the Last Eukaryotic Common Ancestor (LECA),
suggesting that a HGT event from a bacterial source transferred these to the stem
of the eukaryotic lineage. A second set of more rapidly-evolving CDC123 family members
are found primarily in phylogenetically distant amoeboid organisms like Entamoeba, Acanthamoeba, and Naegleria, often in multiple copies (Figure 2). These group with cognates from facultative bacterial symbionts of amoebae, namely
Legionella and giant NCLDVs that infect amoeboid organisms 39] (Figure 2). The complex interplay between Legionella and eukaryotic hosts 40],41] has previously been proposed to have been a conduit for HGT of multiple domains 42],43]. Similarly, transfers between symbionts and viruses sharing the same host cell have
also been documented 44],45],39]. Thus, the distinctive members of the R2K.1 shared by amoeboid eukaryotes and their
symbionts and viruses were likely disseminated via HGT associated with these interactions.
Functional inferences for R2K families based on genome contextual information and
prior experimental results
Based on the conservation of most key catalytic residues or their compensation with
spatially-equivalent residues from elsewhere in the sequence we propose that most
members of the R2K clade are likely to be active enzymes, although in some lineages
this activity may have been lost, most notably in the eukaryotic apicomplexan clade
(Figure 1, Additional File 1). Furthermore, based on the nesting of the R2K clade within the ATP-grasp assemblage,
which primarily catalyzes the formation of peptide-like linkages 16] (Additional file 1), we propose that members of this clade are likely to catalyze similar reactions.
Yeast strains overexpressing CDC123 displayed a second, slightly larger isoform of
CDC123 at low levels 46]. This isoform was suggested to result from an unknown modification to CDC123 and
was linked to its proteasomal degradation 47]. The same work ruled out ubiquitin- and phosphoryl-group additions as potential modifications
resulting in this isoform 47]. In light of the peptide-bond forming activity predicted for the R2K clade ATP-grasp
proteins, we posit that the observed isoform perhaps results from automodification
via serial ligation of amino acids to a particular sidechain or the C-terminus comparable
to the modifications catalyzed by RimK on the ribosomal protein S6 or the TTLs on
tubulins. RimK has been shown to ligate up to fifteen glutamate residues to S6 48],25]; auto-ligation of a comparable number of amino acid residues would be sufficient
to explain the observed larger isoform of CDC123. The interaction networks for various
CDC123 eukaryotic orthologs inferred from high-throughput interactome studies show
an enrichment for multiple proteosomal components 49]. This, together with heat-sensitivity of the CDC123 mutants, suggests that one consequence
of this modification might be to regulate the stability of proteins via the proteasome.
However, it is likely that the CDC123-catalyzed modification has a distinct role in
the context of translation initiation. Physical interaction of CDC123 with GCD11/eIF2?
and the marked decrease in eIF2 complex formation without changes in concentration
of individual eIF2 complex components in the CDC123 null mutants 10] suggest that the modification of particular components might facilitate assembly
of this key translation initiation complex. Similarly, the cell-cycle checkpoint proteins
Chf1/Chf2 8] might also be other targets for modification catalyzed by CDC123.
We then examined the contextual information in the form of conserved gene neighborhoods
and gene fusions of the prokaryotic versions as this has proved to be a useful tool
for deciphering the function of uncharacterized gene products 50],51]. Consequently, we observed that across several phylogenetically distant bacteria,
genes coding for members of the CDC123 (R2K.1) family are embedded within the recently-described
polymorphic toxin loci (Figure 2). Polymorphic toxin systems have been implicated in intra-specific conflicts between
bacteria, acting as the arbiters of “self versus non-self†distinctions between closely-related
organisms 18],52],19],21]. The toxin proteins from these systems are delivered to target cells via a wide range
of secretory systems, which are often genomically-linked to the core loci coding for
the toxin and its cognate immunity protein 18]. Among these secretory systems is T6SS, which utilizes caudate bacteriophage tail-derived
components to inject toxins into target cells 53]. We observed that CDC123 occurs specifically in polymorphic toxin loci with genes
coding for the SUKH domain immunity protein 19] and diagnostic components of the T6SS system including the VgtG, Hcp1, and proteins
with PAAR motifs 18] (Figure 2). As only a subset of polymorphic toxins delivered by T6SS encode a CDC123-like protein,
it is likely to function in a supplementary role, perhaps as a secondary toxin injected
into the target organism or as an auxiliary protein that regulates either the toxin,
the immunity protein, or the secretory apparatus.
The CDC123 family protein found in Legionellae contains extended C-terminal and N-terminal
regions not observed in other CDC123-like proteins (Figure 2, Additional file 1). Legionella secretes several toxins/effectors into its eukaryotic host cell using the Type IV
secretion system (T4SS). The C-terminal region of CDC123 from Legionellae harbors
several of the characteristics known to be important for T4SS delivery such as: 1)
a largely unstructured C-terminal region 54], 2) a conserved hydrophobic residue very close to the C-terminus 54], and 3) a preponderance of both small and polar residues in the ~15 residues upstream
of the hydrophobic residue 55] (Additional file 1). Hence, it is conceivable the Legionella CDC123 is secreted via the T4SS as an effector into the host eukaryotic cell. Thus,
the evidence from the two distinct sets of bacterial members of the CDC123 family
point in the direction of functioning as a secreted toxin or auxiliary factors of
toxin systems, which might modify proteins with peptide tags by means of their peptide
ligase activity. Given the second set of eukaryotic and NCLDV CDC123 homologs are
specifically related to the Legionella versions, it is likely that these perform functions similar to the former and different
from the classical CDC123 translation regulators referred to above. Their presence,
often as multiple paralogous copies (unlike the single-copy classical CDC123 versions)
across phylogenetically distant amoeboid eukaryotes (Additional File 1), raises the possibility that they modify cytoskeletal proteins associated with the
amoeboid cellular morphology, such as components of the actin-based cytoskeleton.
This might parallel the extensive modification of tubulin by peptide tags, ranging
from a single tyrosine to long polyglutamyl or polyglycinyl chains, catalyzed by multiple
ATP-grasp ligases 37],38],56],57]. Thus, such cytoskeletal modifications could be utilized by both the amoeboid organisms
and their symbionts/parasites in facilitating formation of intracellular structures
conducive to their lifestyle.
We observed operonic connections between genes of the R2K.3 family and those coding
for multiple GCN5-like acetyltransferase (GNAT) domains in several actinobacteria
of the Streptomyces lineage, the chloroflexi Herpetosiphon, and the cyanobacterium Acaryochloris (Figure 2). In certain firmicutes and the actinobacteria, genes for the R2K.3 and R2K.2 families
were linked together in the same operon (Figure 2). The operonic linkage of genes for distinct ATP-grasp peptide ligases or unrelated
ligase domains, such as those of the COOH-NH2 ligase or GNAT superfamilies, have been
previously observed in multiple instances 16]. Such linked peptide ligases often catalyze successive peptide ligations with distinct
moieties in the biosynthesis of peptide-derived secondary metabolites like antibiotics
and siderophores, storage polypeptides like cyanophycin, peptidoglycan, teichuronopeptides,
the O-antigen, and cofactors like glutathione 58],25]. Hence, we posit that the R2K.2 and R2K.3 families catalyze peptide ligation, which
might be further followed by action of the second ligase or capped by an acyl group
added by the associated GNAT protein. In certain firmicutes, the linked genes for
the R2K.2 and R2K.3 family proteins sandwich a third gene coding for an ADP-ribosylglycohydrolase
(ARG) (Figure 2). ARGs catalyze the hydrolysis of glycosidic bonds to remove ADP-ribose moieties
conjugated to side-chains of particular residues in proteins by ADP-ribosyltranferases
59],60]. This linkage suggests that, like the ARG, the peptide ligase action of R2K.2 and
R2K.3 enzymes is likely to target proteins. As there are no other linked genes in
these neighborhoods, the identity of their target proteins remain elusive. Nevertheless,
given that at least the R2K.2 family is found in several caudate bacteriophages infecting
phylogenetically-distant bacteria (Additional File 1), it might modify specific host proteins, analogous to the ADP-ribose modification
of the same by phage enzymes 61]-63]. Conversely, even as phage-derived proteins are occasionally redeployed by the host
against other viruses 64], it is possible that the bacterial versions are deployed against proteins encoded
by invasive operons. This proposal is also consistent with the sporadic distribution
of these families indicative of HGT and gene-loss, which is similar to that of other
families of proteins implicated in providing specific selective advantage in biological
conflicts 65],66].
General conclusions
We present the discovery of a novel clade of ATP-grasp enzymes, the R2K clade, which
includes the conserved eukaryotic protein CDC123. We show that this clade displays
certain aberrant features hitherto not encountered in other members of the ATP-grasp
superfamily. Nevertheless, the weight of the evidence suggests that they belong to
the vast assemblage of ligases catalyzing formation of peptide bonds or similar linkages
in the biosynthesis of a variety of compounds and also in the peptide-tag-modification
of target proteins. We propose that the classical CDC123 family is likely to modify
proteins, including possibly components of the eukaryotic eIF2 translation initiation
complex. Importantly, we show that the CDC123 family had its origins in bacteria where
it appears to have diversified first along with the two other families of the R2K
clade. The bacterial CDC123 proteins are of two distinct types, one specifically associated
with T6SS-delivered polymorphic toxin systems and the other probably functioning as
effectors directed at amoeboid eukaryotic hosts. Similarly, the R2K.2 and R2K.3 families
are also proposed to participate in biological conflicts, probably between bacteriophages
and their hosts. Thus, our findings not only help predict an unexpected biochemical
function for a poorly understood translation initiation factor but also help trace
its origin back to bacterial conflict systems, where it might have been deployed as
a toxin in intergenomic/interorganismal conflicts 22],21].
Previously, several key components of the eukaryotic protein modification and signaling
systems, such as the ADP-ribosyltransferases, DOT1-like protein methyltransferases,
and Fic/Doc-like protein AMPylating enzymes, have been traced to polymorphic toxin-
or related host targeting effector-systems of endosymbiotic bacteria 67],18]. CDC123 joins these as a potential protein modification system that was recruited
from a bacterial effector. This observation adds one more piece of evidence to the
recently proposed hypothesis that effectors from the bacterial endosymbionts of the
stem eukaryotes played a fundamental role in the emergence of the characteristically
eukaryotic regulatory systems and sub-cellular structures 21]. Moreover, the diversification of the R2K clade in bacteria and their phages also
adds support to the hypothesis that the exchange of a common set of protein- and nucleic-acid-modifying
enzymatic effectors between disparate bacterial conflict systems helped in their extensive
diversification. Representatives of this pool of enzymes were repeatedly taken up
by eukaryotes and used as components of novel regulatory systems.
Methods
Iterative sequence-profile and HMM searches were performed using the PSI-BLAST 68] and JACKHMMER web utilities (http://hmmer.janelia.org/search/jackhmmer), respectively. Queries were run against the non-redundant (nr) protein database
of the National Center for Biotechnology Information (NCBI). Profile-profile comparisons
were performed using the HHpred program 69]. Multiple sequence alignments were constructed using the MUSCLE alignment program
70] followed by manual adjustment as determined by high-scoring pairs detailed in homology
search results and alignment with experimentally-elucidated protein structures. Alignment
secondary structure predictions were performed with the JPred program 71]. Gene neighborhoods were extracted from PTT and GenBank files (downloadable from
the NCBI ftp server) using Perl scripts. Sequence-based homology clustering of all
proteins determined to belong to the R2K assemblage and proteins encoded in the recovered
gene neighborhoods was performed with the BLASTCLUST program (http://ftp.ncbi.nih.gov/blast/documents/blastclust.html) using empirically-determined scoring and length threshold values. Visualization
and manipulation of protein structure was accomplished using the PyMol program (http://www.pymol.org), structure similarity searches were performed using DaliLite 72]. Phylogenetic trees were constructed using the maximum likelihood method as implemented
by the PhyML program 73].